Glycan profiling is a technique that involves the comprehensive analysis and characterization of the glycan structures present in a biological sample. To determine their role in biological processes, a detailed analysis of the glycans present in a sample, including their identification and quantification, is necessary. Glycans play a role in modulating protein function, cell signaling, and immune responses. Therapeutic strategy development and diagnostic tool creation depend on accurate determination of glycan structure and abundance.
Glycan profiling or glycan analysis describes the mass spectrometric characterization of released glycans in a sample. The process of glycan profiling is complex: there is no template for glycan synthesis, so a glycoprotein or glycolipid will have a distribution of glycoforms and this distribution is dependent on the type of cell, the stage of differentiation and the cell environment. Glycans are released enzymatically or chemically from the glycoprotein or glycolipid and, before analysis, this complex mixture is purified to remove salts and detergents, which will reduce ionization efficiency. The glycans are then derivatised, to stabilize acid-labile residues and/or to add chromophores or mass tags for detection and/or sensitivity. The glycans are separated using normal phase liquid chromatography (hydrophilic interaction chromatography) due to their polarity and are then introduced into the detector. At the detector, the eluting glycans are identified according to their accurate mass and fragmentation pattern. The precise retention time allows tentative structural assignments using known retention units and the fragmentation spectrum can be compared with an internal or online database to confirm the structure. The current state of the art in glycan profiling will not allow the method to run unsupervised, due to the lack of a library of structures for comparison. Manual confirmation is required to identify novel structures, many of which are biologically active. Glycan profiling is used in the diagnosis of diseases because changes in glycan patterns are mechanistically involved in disease and these glycan patterns can be used as biomarkers in early detection of diseases such as cancer, monitoring of inflammation or quality control in biopharmaceutical production.
Fig. 1 Overview of the workflow for the structural analysis of N-linked glycans.1,2
Glycan profiling is widely used in biomedical research from basic biology to translational research including clinical diagnostics and drug development. One of the main uses of glycan profiling is to elucidate and decipher the information encoded in the complex and rich "glycan code". Glycans are dynamic, highly modifiable and sensitive to cellular signals and environmental changes. Glycans can function as a mediator for many proteins that fold, adhere to the cell surface or mediate immune regulation and pathogen recognition. Glycosylation changes have been associated with the progression of various diseases such as cancer, autoimmune and infectious diseases. Glycan profiling enables the identification of disease specific glycan biomarkers and can help understand the pathophysiological mechanism of disease. Glycan profiling is also an important application in biopharmaceutical development, especially in the production and quality control of protein therapeutics.
The number of glycan profiling technologies is increasing. HPLC and HILIC are the most commonly used methods for glycan profiling due to their robustness and high-resolution separation. These methods separate glycans on the basis of their hydrophilicity and are used in combination with refractive index or fluorescence detection for sensitive glycan profiling. CE has been recently demonstrated to be a useful technique for glycan separation. CE is based on the differential migration of glycans in an electric field and provides rapid and efficient separation of glycans. MS has become the most widely used analytical technique for glycan profiling. MALDI and ESI are the two MS methods that have been used most often for glycan profiling. MS-based glycan profiling allows for the detection of glycans in complex biological samples, and can provide compositional information as well as fine structural information, such as linkage position and anomeric configuration. NMR is another important technique that has been used for glycan conformational analysis. NMR can provide information about the conformation and dynamics of glycans, which is important for understanding their biological functions. Lectin arrays and glycan microarrays have been developed for high-throughput glycan profiling. These arrays allow for the rapid screening of glycan-protein interactions and have been used for the discovery of novel glycan biomarkers.
HPLC is one of the most widely used and versatile techniques in glycomics. HPLC is based on the differential partitioning of analytes in a complex mixture (e.g. a glycan mixture) between a mobile phase and a stationary phase. Mass spectrometry, one of the most commonly used techniques to determine glycan composition, often has difficulty distinguishing isomeric glycans. HPLC can separate glycans that would be otherwise indistinguishable by mass spectrometry, as glycans will interact with other glycans as well as stationary phases differently based on their physicochemical properties (e.g. charge, size, hydrophilicity). The analytes in the mobile phase will be retained by the stationary phase to differing extents depending on their affinity to the stationary phase. The retention time of each analyte can then be used to identify and quantify that analyte. HPLC can be performed for glycan analysis using HILIC, normal phase chromatography, or reversed phase chromatography after derivatization. All three can be fine-tuned to achieve selectivity towards specific glycan classes. HPLC is often coupled with MS or fluorescence detection for the relative quantification of glycans in a sample. The resulting chromatogram can provide a unique fingerprint of the glycome of a sample, and the retention time of each peak can be used to infer structural information about the glycans present. HPLC can also be used to separate glycan isomers that may be indistinguishable by MS alone. HPLC is often used for relative quantification of glycans across multiple samples, as it is reproducible, amenable to automation and a "workhorse" method. This makes it particularly useful for applications such as biomarker discovery, biopharmaceutical batch release testing, and monitoring changes in glycan structures in response to disease or other conditions.
Like all chromatography, HPLC-based glycan separation relies on partitioning of each analyte between a liquid mobile phase and a solid stationary phase. The exact mechanism by which this occurs can be adsorption, size exclusion, ion exchange or affinity, and depends on the chemistry of the column used. Hydrophilic interaction liquid chromatography is commonly used for glycan profiling since carbohydrates are very polar, and under the high organic, low aqueous conditions used will partition onto a polar column such as bare silica or amide-bonded silica and be eluted as the aqueous content of the mobile phase is increased. Glycans are thus separated based largely on size and charge, with the larger, more highly sialylated molecules tending to elute later due to stronger hydrogen-bonding. Normal-phase chromatography is similar but the silica columns are used with purely organic eluents, and can better resolve isomeric glycans based on small differences in the orientation of hydroxyl groups. Reversed-phase chromatography, on the other hand, is based on derivatising the glycans with a hydrophobic tag such as 2-aminobenzamide or procainamide, which can then be separated on a C18 non-polar stationary phase by the combined effects of size, charge, and hydrophobicity of the tag. The separation is performed using a high pressure pump to drive a precisely-controlled gradient of solvents, so as to ensure reproducible retention times. The output from a column is typically the result of fluorometric labelling, in which the derivatisation introduces a fluorescent moiety which is highly sensitive to excitation, and so can be photometrically quantified even at picomolar levels. A pump, injector, column, detector, and data processor can function as an integrated system in which the temperature, flow, and gradient reproducibility are critical to the consistency of the generated chromatographic profiles, which separate the glycans in a sample according to their physicochemical properties, with sufficient resolution to distinguish these structures for comparative purposes.
Major advantages of HPLC for glycan profiling include its reproducibility and quantitative potential. The retention time can be standardised between laboratories with the use of retention unit indices and may be matched to curated libraries, allowing structural annotation without the need for MS analysis of every sample. Coupling to fluorescence detection provides an extremely linear response over several orders of magnitude, allowing accurate relative quantification of individual glycans between cohorts. The instrumentation is ubiquitous, rugged and easily automated, allowing for high throughput screening of clinical samples or biopharmaceutical batches. The resolution of isomeric structures, in particular linkage isomers of sialic acid or branching isomers of complex N-glycans, is usually better than what is obtained with intact mass analysis, especially when using specialised stationary phases such as amide-bonded or porous graphitised carbon columns. On the other hand, this method is limited by long analysis times (hours per sample for complex glycomes, unfeasible if hundreds of samples have to be processed) and limited sample throughput (derivatisation, which is often variable and can result in differential recovery of certain glycans, is required before analysis), detection sensitivity (fluorescence-detection sensitivity is excellent but requires labelling; incompletely labelled species may be missed or underrepresented, and native glycans with no chromophores are not directly detectable), column lifetime (glycans, in particular sialylated or sulfated species, tend to bind irreversibly to the stationary phase, with eventual peak tailing, loss of resolution, and column death) and absolute quantification (different glycan structures have different response factors, and calibration against a pure standard is required, but such reference materials are not available for all structures). Finally, the data obtained by HPLC only include retention time, and confident assignment of novel structures requires orthogonal confirmation by MS or NMR, which complicates the workflow and increases cost.
Actual glycomics applications have many variations from analytical batch quality control to structural elucidation of biologically active glycans. HPLC is used for quality control of biopharmaceutical antibody N-glycans, as reproducibility of effector functions must be demonstrated; the amount of each fucosylated, afucosylated and sialylated glycan is determined and compared with limits of acceptance (test range), and if these parameters are not met, the process must be investigated. Serum glycan profiles in oncology, such as glycans released from N-glycans and separated by HPLC after fluorescence labelling, have shown patterns of increased branching and sialylation in metastatic disease, which is associated with worse outcomes. The technique has been used in profiling the changes of O-glycans from mucinous proteins, where a universal enzyme for release is not available and hence mass spectrometric analysis can be difficult; using chemical release followed by HPLC separation to distinguish truncation patterns has enabled profiling of tumor-associated antigens. For example, clinical laboratories use HPLC to separate and quantify the glycoforms of carbohydrate-deficient transferrin, which are biomarkers of chronic alcohol consumption. HPLC has also been used for glycoengineering, or screening glycoforms produced by cells; as the process of making glycoproteins in engineered cell lines can be adjusted by knocking out or overexpressing glycosyltransferases, it is necessary to quickly assess the glycan distribution after such changes are made, which HPLC is suited for. Separations on porous graphitised carbon have been extensively used for resolving sialic acid linkage isomers, which can be used to understand which linkages pathogens have binding specificity for. Although mass spectrometry is often used for structural elucidation, HPLC is still used extensively because of its quantitative nature and relative robustness, which is necessary for monitoring diseases over a period of time, where minor changes in relative abundance can become detectable and thus significant for early detection or response to treatment. By providing reproducible and quantifiable glycome profiles, HPLC serves as a translation bridge between discovery applications using mass spectrometry and the clinical use of glycomic information.
Mass spectrometry (MS) has revolutionized glycan analysis from an approximate chromatographic endeavor into a quantitative and information-rich analysis method, allowing the identification of structural information not accessible from other analytical platforms. Its popularity as an analytical method is due to its direct ionization of intact oligosaccharides, instead of derivatized crystalline forms as was previously necessary, allowing labile modifications (e.g. sialic acids, fucose) that can be lost during the chromatography to be detected. In contrast to HPLC, which separates molecules by their physicochemical properties en masse, MS interrogates each molecule in turn, measuring the accurate mass to determine the elemental composition, and tandem MS fragmentation to determine connectivities and stereochemistry. In terms of soft-ionization methods, two techniques are particularly popular: matrix-assisted laser desorption ionisation (MALDI), which results in predominantly singly charged ions, and electrospray ionisation (ESI), which results in multiply charged ions and which is easily coupled to online chromatography. MALDI, while primarily used for rapid fingerprinting, is most often used for permethylated glycans, and ESI is required for native glycans whose labile acidic groups would otherwise be lost under vacuum. This is one distinction between the two techniques, as MALDI is used primarily for discovery-based profiling while ESI-LC-MS has better isomeric resolution and is used for definite structural assignments. Hybrid approaches are also common, such as using MALDI-TOF for composition analysis and ESI-FTICR for more in-depth structural validation. The data obtained from such analyses can now be used for not only identification but also functional interpretation and correlation to disease states, biopharmaceutical function, and immune recognition, for example. MS analysis has now become a bottleneck, with the development of various algorithms to interpret the data by matching the spectra obtained experimentally with the theoretical fragmentation patterns of all possible glycans.
Mass spectrometry (MS) is used for identification, quantification, and structural analysis of molecules. The standard procedure of MS-based glycan profiling starts with the ionization of glycans into ions, followed by separation and detection of these ions in a mass spectrometer based on their mass-to-charge ratio (m/z). The detected ions are then used to form a mass spectrum for further structural interpretation. The mass spectra contain information on glycan structures in the sample that can be used for composition, sequence, and linkage analysis. MS offers high sensitivity and specificity for glycan analysis. The separation and detection of ions are performed by various techniques, with the selection depending on the analytes and requirements of the analysis. Electrospray ionization (ESI) has been used for intact native glycans without dissociation of labile acidic groups, while permethylated glycans were effectively profiled using vacuum MALDI MS and tandem MS. During ESI, the analytes are dissolved in a solvent and passed through a charged capillary to create ions. In MALDI, the analytes are mixed with a matrix and irradiated with a laser beam to generate ions. The ions produced are then introduced into the mass spectrometer, where they are separated and detected based on their m/z ratios. The mass spectrometer typically consists of an ionization source, a mass analyzer, and a detector. The ionization source ionizes the analytes, the mass analyzer separates the ions based on their m/z, and the detector detects the separated ions. The detected ions are then used to create a mass spectrum that is used for structural interpretation of glycans. MS techniques can be categorized into various types based on the ionization method, mass analyzer used, and the detection method employed.
MALDI-time-of-flight (MALDI-TOF) is often used for quick compositional analyses; spectra are obtained directly from a dried sample spot, and permethylated glycans of both neutral and acidic species can be high-throughput screened in a matter of minutes. It is the method of choice for glycomic screening and quantification of large numbers of samples, though the degree of fragmentation of the sialic acids is not controlled. Unspecific sialic acid cleavage can be prevented by on target esterification or by an ESI-based method. ESI ionisation, often coupled with hydrophilic interaction liquid chromatography (HILIC), is considered the current standard in native glycan analysis. The online separation based on polarity prior to MS detection is particularly helpful in resolving isomers. In addition, source conditions can be adjusted to be very mild, so labile modifications can be preserved. Another chromatographic separation, using porous graphitised carbon (PGC), provides orthogonal selectivity, by means of shape and hydrophobicity, and is particularly good at separating linkage isomers that co-elute on amide columns. Glycans modified with reductively aminated tags can be separated by reversed-phase liquid chromatography (RP-LC), as the tags confer hydrophobicity, with the resulting separation based on size and charge. These approaches are also differentiated by tandem mass spectrometry strategies; collision-induced dissociation (CID) gives a high yield of glycosidic bond cleavages, which can be used to determine glycan sequence, while electron-transfer dissociation (ETD) causes cross-ring fragmentation, which can be used to determine linkage, an important property to determine, for example, α2,3- versus α2,6-sialylation. Multistage tandem MS, where fragment ions are themselves isolated and fragmented, can be used for gas-phase dissection of complex isomeric mixtures, for example, to distinguish between antennae fucosylation patterns. In-source fragmentation can be introduced, by increasing the cone voltage, to pseudo-hydrolyse polysaccharides, and determine the repeating unit composition, without chemical hydrolysis.
The principal advantage of mass spectrometry is in resolving isomeric species: chromatographic separation alone cannot distinguish structures with the same overall mass and elution properties. By fragmenting glycans in the gas phase, MS measures their connectivity, specifically the linkage of each monosaccharide, and can differentiate linear from branched isomers, and in many cases the specific type of linkage (α or β, 1–2, 1–3, 1–4, 1–6, etc.) as well, based on cross-ring cleavage ions that "remember" their origin. After permethylation, where mass tags at each oxygen allow identification of the linkages, MS can unambiguously determine the topology of a glycan, without any structural information a priori. Elemental composition is further verified with high mass accuracy (to a few ppm), achievable with high-resolution analysers such as FTICR and Orbitrap, that determines the formula without ambiguity and can immediately discard any putative structures that are chemically not possible. The sensitivity of MS extends to femtomole quantities of glycan, allowing analysis from limited sample material such as a needle biopsy, or a finite number of cells, necessary for personalized medicine applications. The speed with which MS can be performed is also a great advantage for glycomic and glycoproteomic studies; a MALDI glycomic profile is acquired within minutes, and LC-MS can be done in under an hour, allowing both high-throughput cohort studies as well as bioprocess monitoring. The multiplexing ability of MS means that even complex structural diversity can be interrogated at the same time, as neutral, sialylated, sulfated, and phosphorylated glycans can be detected in a single acquisition; by comparison, resolving these species on chromatography would take at least four chromatographic runs. For biologics, MS can be used to confirm glycan occupancy at each site, ensuring batch-to-batch consistency, or detect rare glycans that might be immunogenic.
Chromatography and mass spectrometry are the major tools for analyzing glycans but other techniques can provide additional selectivity, structural information or increased sample throughput. Capillary electrophoresis (CE) provides a separation mechanism that is independent from the liquid chromatography (LC) separation, which can be useful for resolving glycan isomers, even on very high resolution columns, if they have different charge states. Nuclear magnetic resonance (NMR) spectroscopy, although limited by a higher sample amount requirement, provides the only direct measurement of the anomeric state and glycosidic bond geometry. Recent developments in microfluidic devices have increased the applicability of CE. The main advantage is that the small size of these devices decreases sample amounts required and necessary analysis times to the picoliter and minute range. Ion mobility spectrometry (IMS) has also been added to mass spectrometric analysis to separate molecules in the gas phase prior to mass analysis, providing additional information for structure assignment. In general, each of these separation methods can provide unique information and advantages: CE is faster and more high-resolution based on charge differences; NMR is direct; and microfluidics/IMS will likely move towards single-cell detection and real-time glycan analysis. It is important to note that these techniques do not replace the current use of LC-MS. Instead, these new tools can be used for more specific needs, such as identifying linkage isomers or confirming absolute stereochemistry, as well as spatially mapping glycans within cells and tissues that are not accessible by bulk analysis. As more techniques are being used for glycomic analysis, there is a need to couple multiple platforms to achieve a more comprehensive glycan characterization.
Capillary electrophoresis (CE) is a high-resolution method of electrophoretic separation within a narrow fused-silica capillary in response to an electric field. The technique is used to separate and analyse charged molecules. It provides higher resolution separations than traditional high-performance liquid chromatography (HPLC). CE is carried out in a narrow capillary by applying an electric field to a charged electrolyte solution, which causes an electro-osmotic flow of the solution. The electro-osmotic flow is caused by positively-charged salt ions in the solution moving along the negatively-charged walls of the capillary, dragging the rest of the solution with them. A charged sample is injected at one end, and the components of the sample are separated by their electrophoretic mobility, which is a function of the electric field, the charge of the molecule, the size of the molecule, and interactions between the molecule and the other molecules in the solution. Charged molecules move at different speeds depending on their charge, with positively-charged molecules moving more quickly toward the negative end of the capillary, and negatively-charged molecules moving more slowly. Neutral species are carried with the bulk flow of the solution. The separated molecules are detected by UV-Vis or fluorescence methods, with the resulting electropherogram providing information about the number of compounds in the mixture and their relative amounts. CE is used for glycan profiling because it has high sensitivity, rapid analysis times and low sample consumption. It is used for analysis of complex mixtures, and has been used in biochemistry, pharmacology and clinical research. Multi-capillary formats have been developed, increasing the throughput, and the sensitivity of CE for glycan profiling has been improved by combining it with laser-induced fluorescence (CE-LIF).
Nuclear Magnetic Resonance (NMR) spectroscopy is a non-destructive analytical technique that can provide detailed information about the structure, dynamics, and interactions of glycans. NMR involves placing a sample in a strong magnetic field and applying radiofrequency pulses that excite the nuclei of certain atoms, such as hydrogen or carbon. As the excited nuclei relax back to their equilibrium state, they emit signals that are detected and analyzed. The resulting NMR spectra can provide information about the chemical environment of the nuclei, which can be used to deduce the structure of the glycan, including its monosaccharide composition, linkage positions, and anomeric configurations. NMR is particularly useful for studying the conformation and dynamics of glycans in solution, as it can provide information about their three-dimensional structures and interactions with other molecules. NMR is complementary to MS, as it provides information about the topology and flexibility of glycans, rather than just their composition and sequence. NMR spectroscopy is an important tool for glycan profiling as it can provide detailed information about the structure and conformation of glycans. NMR generally requires relatively large sample amounts and longer analysis times compared to other techniques like CE or MS.
Emerging technologies and future directions As new technologies continue to emerge, they expand the limits of what is possible in glycan profiling. Microfluidics and lab-on-a-chip technologies, for example, are enabling miniaturized and automated platforms for glycan analysis. By integrating multiple analytical steps (e.g., enzymatic digestion, purification, labeling) into a single microfluidic chip, these platforms can dramatically reduce sample consumption, analysis time, and improve reproducibility and throughput. Glycan arrays and shotgun glycomics are also being developed for high-throughput screening of glycan-protein interactions. Glycan arrays involve immobilizing glycans on solid surfaces and probing them with glycan-binding proteins (GBPs) or antibodies to identify glycan-binding specificities. Shotgun glycomics, on the other hand, involves isolating glycans from biological samples and profiling them using advanced MS techniques to analyze glycan diversity in complex mixtures. In addition, metabolic labeling and photo-crosslinking technologies are being developed to study glycan dynamics and interactions in real-time within living cells. These emerging technologies, along with advances in machine learning and big data analytics, are transforming glycan profiling into a more efficient, precise, and insightful endeavor. They hold great promise for accelerating discoveries in glycobiology and driving the development of novel glycan-based therapeutics and diagnostics.
Glycan profiling is being used as a bridge from structural glycobiology to clinical translation and biopharmaceutical development. The role of glycan profiling is varied, where its use is applied across disciplines to decipher the carbohydrate code as to uncover the molecular signatures in data which are opaque in proteomic and genomic data. In protein therapeutics manufacture, for example, it is used to ensure batch-to-batch consistency of protein batches by demonstrating that the glycoforms known to be associated with effector function and pharmacokinetics remain within predetermined specification limits. In oncology, for example, serum or tissue glycomic profiling screens for patterns of glycan branching and sialylation which are associated with malignant progression, metastatic potential and therapeutic resistance. These can then be used as non-invasive biomarkers for patient stratification. For infectious disease discovery, glycan profiling is applied to chart viral envelope glycosylation to aid vaccine design by revealing antigenic glycan surfaces that are variably exposed in circulating strains.
The clinical efficacy of protein therapeutics is often largely determined by glycosylation, impacting on potency, serum half-life, immunogenicity, and effector function. Monoclonal antibodies are one example where the absence of glycosylation is known to abrogate function. The Fc portion of the antibody features a conserved asparagine-linked glycan. Variation in this glycan's composition affects binding to Fcγ receptors and complement proteins. The afucosylated form of the glycan in particular results in increased affinity to activating FcγRIIIa receptors, and therefore improved antibody-dependent cellular cytotoxicity. This is the intended design of second generation antibodies used in hematological malignancies where immune system targeting is critical, while the core-fucosylated version inhibits FcγRIIIa binding, making it a useful design for antibodies that function primarily through direct signaling inhibition. In addition to fucosylation, other modifications such as bisecting GlcNAc and terminal sialic acid also have effects on receptor binding and anti-inflammatory activity, and the presence of high-mannose glycans can increase receptor-mediated clearance by the mannose receptor, and is sometimes desirable to reduce half-life. The erythropoiesis stimulating protein's high sialic acid content is necessary to prevent asialoglycoprotein receptor uptake in the liver and maintain a long serum half-life. As such, glycan analysis is typically a required quality control step in the manufacturing process and can even be a criterion for regulatory approval, as the FDA typically requires manufacturers to demonstrate batch consistency.
The utility of glycan profiling as a diagnostic and prognostic tool in disease states is rooted in the fact that cells and tissues remanufacture their glycomes in predictable patterns during development and disease progression. Glycan signatures arising in this manner can be interrogated in accessible biofluids or tissue specimens. For example, cancerous transformation is associated with incomplete synthesis of complex glycans, leading to the exposure of truncated precursors that act as decoy ligands for inhibitory lectins present on natural killer cells. As a result, tumor cells are able to avoid innate immune recognition. These same truncated glycans are shed into circulation, and the profile of N- and O-glycans present on serum glycoproteins can be used to identify patients at risk for metastasis and resistance to therapy. Changes in the degree of N-glycan branching and in the level of hypersialylation are also associated with changes in growth-factor receptor signaling and vascular extravasation, and can provide prognostic information in concert with genomic markers. Aberrant fucosylation and sialylation of acute-phase proteins in inflammatory disease can also be monitored over time to assess disease activity and response to immunomodulatory therapies, often before the disease is otherwise detectable. Infectious diseases produce distinct glycan signatures on the surfaces of pathogens as well as on host response proteins, and the glycomic maps of these patterns can be used to inform vaccine design by determining which carbohydrate epitopes are exposed and which are masked. Genetic disorders of glycosylation result in unique glycomic signatures that can be used to aid in diagnosis.
One of the primary challenges in glycan analysis is the fundamental complexity of carbohydrates themselves, which, coupled with the underdeveloped state of analytical infrastructure, has hindered the translation of glycan profiling from bench to bedside. The issue of isomeric redundancy is central; structurally distinct glycans that possess the same mass and composition but differ in linkage type, branching order, or anomeric state will produce indistinguishable signatures when analyzed by a single method. This redundancy necessitates orthogonal confirmation, but integrating multiple analytical approaches invariably increases the consumption of limited samples, prolongs analysis time, and escalates costs. Additionally, the process of sample preparation and purification itself introduces significant losses and biases; the efficiencies of enzymatic release of N- and O-glycans differ, chemical release methods may result in desialylation, and each purification step can lead to selective losses that compromise quantitative accuracy.
Resolution and sensitivity form the x- and y-axis, respectively, where the upper limits of current glycan profiling technologies have been defined. Sensitivity is limited by the intrinsic ionisation efficiency of glycans that depends on their charge, size, and presence of labile residues. Sialylated glycans suffer losses through in-source fragmentation, while uncharged glycans have poor ionisation efficiency and require derivatisation, lowering the throughput and introducing bias to the measurements. Detection limits, albeit quite low for mass spectrometric detection, are generally not low enough to capture all glycoforms, including the low-abundance subpopulations which can be biologically significant in tissue microenvironments. Resolution is limited by isomeric crowding. Glycans with the same mass and different linkage geometry have co-elution and isobaric fragmentation spectra in chromatographic separations, and so they cannot be assigned without orthogonal confirmation. CE isomeric separation is better than chromatographic techniques, but requires charge, and CE peak reproducibility has suffered from adsorption to capillary walls. LC is robust, but linkage isomers cannot be separated without loss of speed and increase in cost of chromatographic separation. Multistage tandem MS is powerful, but results in forests of fragment ions where diagnostic peaks can be obscured by noise and require expert interpretation. New methods are being developed to address some of these limitations, for example by gas-phase ion mobility which separates ions by their shape and collisional cross-section, enabling differentiation of α2,3- versus α2,6-sialylated isomers without the need for derivatisation.
Automation is crucial for translating glycomics into clinical diagnostics, allowing the high sample throughput necessary for the validation of biomarkers and the release of biopharmaceutical batches. Multi-step processes including protein denaturation, glycan release and purification, fluorescent labelling, and clean up have been performed in 96-well plates using robotic liquid handling stations, limiting manual hands-on time and maximizing reproducibility while minimizing human error. Multicapillary electrophoresis, with hundreds of capillaries run in parallel, reports glycome data within 30 minutes per sample, an improvement over the time required for sequential LC runs. Automation has also been integrated into ultra-high-performance liquid chromatography instruments, which with the addition of autosamplers and integrated software control, can automatically inject, separate, and acquire data, and intelligent inclusion list-driven tandem mass spectrometry methods can automatically fragment glycan masses of interest. Sample preparation is the remaining bottleneck for full automation; however, solid-supported hydrazide capture and on-filter deglycosylation have been shown to eliminate transfer steps, and end-to-end automation from crude lysate to purified and labelled glycans is possible. Automation in data analysis is also critical; automated annotation of glycan peaks using machine learning algorithms trained on spectral libraries is possible with minimal user input. Outlier samples can be flagged, and relative abundances of glycans across samples or cohorts can be reported. Simplified sample preparation is also needed to accommodate high-throughput glycomic profiling: label-free glycomic analysis methods that omit derivatisation steps can simplify the process, shortening the time and limiting introduced artefacts. Microfluidic devices have been used to pre-concentrate, separate, and detect glycans, with assays only requiring picoliters of sample, however, this comes at the expense of a decreased amount of information that can be obtained with such rapid profiling methods; higher tiered follow-up analyses are needed to reveal glycoform structural detail.
The status of glycan profiling has moved from a method of rare use to a generalizable tool for understanding carbohydrate-based regulation in normal and diseased states. Glycans have been recognized as a repository of non-template encoded orthogonal information to the genome, critically involved in protein folding, cell-cell communication, and overall homeostasis. Technical advances in this field, from chromatographic separations to integrated mass spectrometry and machine learning, have ushered in a new era of analysis from cataloging to predictive biomarker discovery. Present challenges include isomeric resolution, sample processing automation, and standardization of data interpretation between platforms. Future directions in the glycomics field include microfluidic sample processing, gas-phase ion mobility separation, and real-time metabolic labeling approaches. These will likely lead to further miniaturization and decreased cost, allowing for widespread adoption and population-level screening. The end goal of these approaches will be the incorporation of glycomic information into clinical workflows along with genomic and proteomic data to provide a more holistic view of a patient's physiology. Concerted work is needed to standardize reference materials, data processing algorithms, and clinical education.
A typical glycan profiling analysis uses multiple, orthogonal methods. Mass spectrometry has high sensitivity and compositional detail; MALDI is the quickest method for fingerprinting permethylated glycans. Electrospray ionisation in conjunction with LC preserves native structures and allows relative quantification. Fragmentation methods, including CID and ETD, yield sequence and linkage geometry information, which allows for structural assignments with high confidence. HPLC gives a robust separation based on the hydrophilicity and charge of glycans; HILIC separates based on size and polarity; porous graphitised carbon chromatography separates linkage isomers with shape-selective interactions; and reversed phase chromatography coupled with fluorescence detection enables sensitive relative quantitation of fluorescently labelled glycans. Capillary electrophoresis provides orthogonal separation based on charge to mass ratio and can be used to resolve differences in sialic acid linkage that are not resolvable by chromatographic techniques. Nuclear magnetic resonance spectroscopy is the "gold standard" technique for determining anomeric configuration and 3D structure; however, it requires large amounts of sample material. New approaches to glycan profiling such as ion mobility spectrometry and lab-on-a-chip (microfluidic) based approaches are further improving the sensitivity to the level of single cell analysis and dramatically increasing the speed of these methods. Integrated approaches using a tiered approach are common for profiling glycans, such as using lectin arrays or MALDI-TOF for high-throughput screening, LC-MS/MS for comprehensive characterisation, and NMR for validation of individual glycans of interest. Integrated profiling workflows effectively convert individual analytical methods into a single powerful analysis method capable of sufficiently comprehensive, reproducible and rapid mapping of glycan heterogeneity to address biomedical questions and begin clinical translation.
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